Ethers

ABSTRACT

Prostacyclin, its salts, biosynthesis and synthesis thereof, pharmaceutical formulations containing them, and their use in medicine.

This is a divisional of co-pending application Ser. No. 712,788 filed onMar. 18, 1985 which is a continuation of Ser. No. 795,524 filed on May10, 1977 now U.S. Pat. No. 4,539,333.

This invention relates to the extraction, isolation and synthesis of aprostaglandin derivative, its use as a chemical intermediate,formulations containing it, and its use in medicine.

Prostaglandin endoperoxides (PGG₂ and PGH₂) are generated fromarachidonic acid by a membrane-bound cyclo-oxygenase enzyme system,described by Hamberg and Samuelsson (Biochem, Biophys. Acta. 326,448-461, 1974) and are subsequently transformed to PGF₂α, PGE₂, PGD₂, orThromboxane A₂. Thromboxane A₂ shares with the prostaglandinendoperoxides the important biological properties of contracting stripsof rabbit aorta and aggregating blood platelets.

The Applicants have now found that microsomes derived from a variety ofmammalian tissues catalyse the enzymic transformation of theprostaglandin endoperoxides to a prostaglandin derivative (hereinafterreferred to as Prostacyclin which does not contract strips of rabbitaorta, relaxes strips of rabbit coeliac mesenteric and coronaryarteries, has a potent anti-aggregatory action on blood platelets, is astrong vasodilator in whole animals and has other properties describedhereinafter.

Microsomes derived from rabbit or pig blood vessels such as veins andarteries, and rat stomach fundus produce about 80-90% conversion of theprostaglandin endoperoxides. Microsomes derived from rabbit lung tissueand rat pyloric tissue produce 25% conversion of the prostaglandinendoperoxides, whereas those derived from rat kidney, brain, spleen,liver, heart and seminal vesicle tissues produce 5% or less conversionof the prostaglandin endoperoxides.

Prostacyclin is unstable at room temperature in aqueous medium, having ahalf-life of approximately 10 mins., but its anti-aggregatory activitycan be preserved for several days by dissolving the substance in aqueousalkali or in dry acetone and storing at -20° C. On average prostacylinis 10-40 times more potent as an anti-aggregatory agent than PGE₁ and5-20 times more potent than PGD₂, itself a potent inhibitor of plateletaggregation. Prostacyclin also interrupts and reverses the presence ofplatelet aggregation.

Prostacyclin may be prepared biosynthetically by incubating PGG₂ or PGH₂with aortic microsomes in a suitable buffer solution such as Trisbuffer, for approximately 2 minutes at a temperature in the region of22° C. Conversion of the prostaglandin endoperoxides is approximately85%.

Extraction of Prostacyclin is achieved by the addition of cold (0° C.)dry diethyl ether to the incubation mixture. The addition of cold etherstops the enzyme reaction and Prostacyclin enters the ether phase whichmay be separated from the aqueous phase. Evaporation of the ether bystandard techniques such as bubbling nitrogen through the solutionresults in the Prostacyclin being left as a residue which maysubsequently be resuspended in an aqueous solution for furtherexamination or dissolved in anhydrous acetone and stored at atemperature in the region of -20° C. for future use.

The aortic microsomes employed in the incubation mixture may beextracted from pig or rabbit aortas. Aortas may be frozen solidpreferably by dropping them in liquid nitrogen, and then crushed to forma powder which is resuspended in a suitable buffer solution andsubsequently homogenized. The homogenate may then undergo sequentialcentrafugation so as to isolate the microsomal fraction which may beresuspended in deionized water and lyophilized. The incubation mixturewas shown to have an immediate anti-aggregatory effect by monitoring theaggregation of human blood platelets in a Born aggregometer.

Prostacyclin may be prepared using other tissues identified above insubstantially a similar manner. Prostacyclin formed in such anincubation mixture appears to be different from the other products of PGendoperoxides so far described. Its biological properties on theisolated tissues, its instability and its potent anti-aggregatoryactivity show that Prostacyclin is not being PGE₂ or PGF₂α. The presenceof prostaglandin D₂ isomerase in homogenates of several tissues has beendescribed. As prostacyclin is unstable and is a more potentanti-aggregatory agent than PGD₂, it cannot be regarded as PGD₂.Furthermore, PGD₂ isomerase is present in the 100,000 g. supernatant, afraction that did not produce Prostacyclin from PG endoperoxides.Moreover, PGD₂ isomerase needs glutathione as a cofactor and theincubations were carried out in the absence of cofactors. PGE₂, PGF₂αand PGD₂ were not substrates for aortic microsomes and therefore 15-ketoPGs and other products of prostaglandin catabolism could not beconsidered as Prostacyclin. Prostacyclin is also unlikely to be a known15-hydroperoxy PG, firstly because 15-hydroperoxy PGE₂ has a contractileactivity on rabbit aortic strip and secondly the product(s) of thespontaneous decay of Prostacyclin when bio-assayed did not behave likePGE₂, PGF₂α, or PGD₂. As Prostacyclin has an anti-aggrgatory activity itcannot be identical to Thromboxane A₂ or B₂ as they are pro-aggregatorysubstances.

Further studies have shown that Prostacyclin has the chemical structureshown in formula (I) (R is hydrogen) (see Johnson et al.,Prostaglandins, 12/6, 915-928, 1976). ##STR1##

By the present invention we provide the compounds of formula (I) whereinR is hydrogen or a pharmacologically acceptable cation (hereinafterreferred to as Prostacyclin and salts thereof). Salts of Prostacyclininclude alkali metal salts, alkaline earth metal salts and salts oforganic bases.

Salts of Prostacyclin may be synthesized from a compound of formula (II)wherein either or both of Z¹ and Z² is hydrogen or a blocking group suchas acyl, or trialkylsilyl (for example trimethylsilyl). Oxidative attackby iodine or potassium triiodide in the presence of a metal bicarbonateat the 5,6-double bond of a compound of formula (II) with simultaneousor subsequent cyclisation involving the 9-hydroxy group produces acompound of formula (III). Upon treatment with a suitable base such asan organic base or a metal alkoxide, a compound of formula (III) maythen be dehydrohalogenated, resulting in the introduction of a5,6-double bond. This reaction sequence is illustrated in the followingreaction scheme: ##STR2## wherein Y is OH, NHR¹ or OR¹, R¹ being alkylof 1 to 4 carbon atoms or a cation; X is iodo or bromo; and Z¹ and Z²are as defined above.

Included in compounds of formula (IV) are compounds of formula ##STR3##

wherein R is hydrogen or a pharmacologically acceptable cation and eachof Z¹ and Z² is hydrogen or a blocking group.

When Z₁ and/or Z² in formulae (II), (III) and (IV) are blocking groups,the resulting blocked derivatives of the compounds of formula (I) may beconverted to the corresponding compounds of formula (I) by methods knownin the art, for example base hydrolysis.

Prostacyclin salts may be prepared by treating an ester thereof (formula(I): R is alkyl) for example the methyl ester, with a strong base suchas sodium hydroxide in a suitable solvent and lyophilising the resultingreaction mixture. Prostacyclin itself may be conveniently prepared bybase hydrolysis of its corresponding esters or amides in the presence ofan equivalent amount of a caustic alkali in an aqueous alcohol or anaqueous tetrahydrofuran medium, and extracted into an organic solvent atlow temperature.

Prostacyclin and its salts are useful as intermediates in the synthesisof prostaglandin analogues, and exhibit a potent anti-aggregatory actionon blood platelets, and therefore have a particular utility in thetreatment and/or prophylaxis of mammals as anti-thromobtic agents.

They are also useful in mammals, including man, to reduce and controlexcessive gastric secretion, thereby reducing or avoidinggastrointestinal ulcer formation, and accelerating the healing of suchulcers and lesions already present in the gastrointestinal tract.

Prostacyclin and its salts further exhibit vasodilatory action on bloodvessels and therefore have a particular utility as anti-hypertensivesfor the treatment of high blood pressure in mammals, including man.Platelets can be assimilated into the vascular endothelium or evenincorporated into endothelial cells. Biochemical co-operation betweenplatelets and vascular endothelium in the generation of Prostacyclincontributes to the repair of vascular endothelium, and Prostacyclin andits salts have a further utility in the promotion of wound healing inmammals, including man.

Prostacyclin and its salts may be used whenever it is desired to inhibitplatelet aggregation, to reduce the adhesive character of platelets, andto treat or prevent the formation of thrombi in mammals, including man.For example, they may be used in the treatment and prevention ofmyocardial infarcts, in the treatment of peripheral vascular disease totreat and prevent post-operative thrombosis, to promote potency ofvascular grafts following surgery, and to treat complications ofarteriosclerosis and conditions such as atherosclerosis, blood clottingdefects due to lipemia, and other clinical conditions in which theunderlying etiology is associated with lipid imbalance orhyperlipidemia.

They may also be used as additives to blood, blood products, bloodsubstitutes, and other fluids which are used in artificialextra-corporeal circulation and perfusion of isolated body portions,e.g., limbs and organs, whether attached to the original body, detachedand being preserved or prepared for transplant, or attached to a newbody. During these circulations and perfusions, aggregated plateletstend to block the blood vessels and portions of the ciruclationapparatus. This blocking is avoided by the presence of Prostacyclin. Forthis purpose, Prostacyclin or its salts may be added gradually or insingle or multiple portions to the circulating blood, to the blood ofthe donor animal, to the perfused body portion, attached or detached tothe recipient, or to two or all of those at a total steady state dose of0.001 to 10 mg., per liter of circulating fluid. It is especially usefulto use Prostagladin in laboratory animals e.g. cats, dogs, rabbits,monkeys and rats, for these purposes in order to develop new methods andtechniques for organ and limb transplants.

The amount of prostacyclin or a salt thereof required (hereinafterreferred to as the active ingredient) for therapeutic effect will varywith the route of administration. In general a suitable dose for amammal will lie in the range of 0.01 to 200 mg. per kilogram bodyweight,conveniently 0.01 to 10 mg per kilogram.

While it is possible for the active ingredients to be administered asthe raw chemical it is preferable to present it as a pharmaceuticalformulation. Such formulations are preferably non-aqueous andnon-hydroxylic in nature, but alkaline aqueous solutions may be used.Unit doses of a formulation contain between 0.5 mg. and 1.5 g of theactive ingredient.

Such formulations, both for veterinary and for human medical use, of thepresent invention comprise the active ingredient, as above defined,together with one or more acceptable carriers therefor and optionallyother therapeutic ingredients. The carrier(s) must be "acceptable" inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The formulations include those suitable for parenteral (includingsubcutaneous, intramuscular and intravenous) administration which mustof course be sterile.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

According to the present invention there are therefore provided:

(a) the compounds of formula (I) and derivatives thereof blocked in the11- and 15-positions;

(b) the preparation of Prostacyclin comprising:

(i) incubation of microsomes of fresh mammalian tissue with aprostaglandin endoperoxide, and the extraction of prostacyclin from theincubation mixture into an organic solvent; or

(ii) its synthesis as hereinbefore described;

(c) Prostacyclin when obtained by the process described in paragraph(b);

(d) a pharmaceutical formulation containing prostacyclin;

(e) the preparation of such pharmaceutical formulations;

(f) method for the treatment or prophylaxis of thrombosis in a mammal ormammalian tissues, including man, comprising the administration of anon-toxic, prophylactic anti-thrombotic amount of a compound of formula(I).

(g) method for inducing vasodilation in a mammal, including man,comprising the administration of a non-toxic, vasodilatory amount of acompound of formula (I);

(h) method for the prophylaxis or treatment of gastric lesions in amammal, including man comprising the administration of a non-toxic,prophylactic or therapeutic amount of a compound of formula (I);

(i) (5Z)-5,6-didehydro-9-deoxy-6,9α-epoxyprostaglandin-F₁α and its salts

(j) method for the promotion of wound healing in a mammal, includingman, comprising the administration of a non-toxic wound-treatment amountof a compound of formula (I).

The following examples are provided by way of an illustration of thepresent invention and should in no way be construed as constituting alimitation thereof.

EXAMPLE 1 Preparation of Prostacyclin

Pig aortas were stripped of adventitia, snap frozen in liquid nitrogen,crushed into a fine powder, resuspended in 0.05M Tris buffer (pH 7.5)(1:4, w:v) and homogenised at high-speed in a Polytron (KIMENATIC,LUCERNE, SWITZERLAND) homogenizer. The homogenate was centrifuged at1000×g for 15 minutes and the resulting supernatant centrifuged again at10,000×g for 5 minutes. The 10,000×g pellet was discarded, while thepellet obtained after centrifugation of the supernatant at 100,000 g for60 minutes was resuspended in deionized water and lyophilized. Anaverage yield of 150 mg. of aortic microsomal powder (51% protein) per100 g. of aortic tissue was obtained.

Aortic microsomal powder (5 mg) was incubated with 1 μg of prostaglandinendoperoxide (PGG₂ or PGH₂) in 0.05M Tris buffer (pH 7.5) (1 ml.) for 2minutes at 22° C. Enzyme activity was estimated by direct bioassay ofthe incubation mixture. After incubation of 2 minutes at 22° C. allprostaglandin endoperoxide activity was lost indicated by a lack ofcontraction of rabbit aorta strips when assayed by the cascadesuperfusion technique of Vane (Br. J. Pharmac. 23, 369-373, 1964,indicating 100% conversion of PGG₂ or PGH₂.

The Prostacyclin was extracted by the addition of cold dry diethyl ether(1 ml) to the incubation mixture which also stopped the enzymicreaction. The Prostacyclin entered the ether phase, which wassubsequently separated from the aqueous phase. The ether was evaporatedby bubbling nitrogen through it leaving the Prostacyclin which waseither dissolved in ice-cold 0.05M Tris buffer (0.5-1 ml.) andimmediately used for platelet aggregation studies or was dissolved inanhydrous acetone (1 ml.) and stored at -20° C. for future use.

The anti-aggregatory activity of the extracted Prostacyclin disappearedon bioling (15 seconds) or on standing at 22° C. for 20 minutes. Theanti-aggregatory activity of Prostacyclin could be preserved for severaldays by dissolving the substance in dry acetone and storing at -20° C.

By using rabbit aortas in a similar experiment the same results wereobtained.

EXAMPLE 2

Aggregation of platelets in 1 ml. of fresh human platelet rich plasma(PRP) was monitored in a Born aggregometer. An immediateanti-aggregatory effect of the fresh reaction mixture of Example 1 ofaortic microsomes with PGH₂ or PGG₂ was observed. The lowestanti-aggregatory concentration was obtained with samples containing0.5-5 ng. Prostacyclin/ml.

The activity disappeared after leaving the incubation mixture for 20minutes at 22° C. or by bioling for 15 seconds. Aortic microsomes alone(50 μg/ml) could induce aggregation in some PRP. The products ofspontaneous degradation of PGG₂ (100 ng/ml) had no anti-aggregatoryactivity.

Diethyl ether extracts of Prostacyclin also inhibited plateletaggregation induced by arachidonic acid and PGG₂. The effectiveanti-aggregatory concentration of Prostacyclin after storate in etherwas from 1 to 10 ng/ml.

EXAMPLE 3

Prostacyclin was injected intravenously and intra-aortically intonormotensive anaethetised rats and the blood pressure and heart raterecorded. Results showed Prostacyclin to be a powerful vasodepressorhaving 5 times the potency of PGE₂.

EXAMPLE 4

Prostacyclin was found to relax spirally cut strips of coronary arteryfrom the ox. The effect is dose dependent and relaxation can be seenwith doses as low as 20 nanograms per 5 ml bath. In isolated hearts fromrabbits perfused at constant flow rate by the Langendorf technique,Prostacyclin produced coronary vasodilation.

Prostacyclin relaxed strips of coeliac and mesenteric arteries, but wasless potent than PGE₂.

EXAMPLE 5

Prostacyclin was shown to inhibit the formation of gasteric lesionsinduced in rats by indomethacin at doses from 62.5 to 250 μg uponsubcutaneous injection.

EXAMPLE 6

A stirred solution of PGF₂α methyl ester (50 mg) in ether (1 ml) wastreated with sodium bicarbonate (115.0 mg; 10 molecular equivalents) andwater (1 ml) and then dropwise during 2 hours with aqueous potassiumtriiodide (0.7 molar; 0.261 ml). After stirring overnight, the reactionmixture was shaken with ether and aqueous sodium thiosulphate; theetheral phase was separated, washed with water, dried with magnesiumsulphate, and evaporated to leave a yellow gum of5ξ-iodo-9-deoxy-6ξ,9α-epoxyprostaglandin F₁α methyl ester.

A solution of 5ξ-iodo-9-deoxy-6ξ,9α-epoxyprostaglandin F₁α methyl ester(100 mg.) in methanolic sodium methoxide prepared from sodium (46 mg.)and dry methanol (0.70 ml.) was set aside under dry nitrogen for 5hours, then freed from solvent in high vacuum. The residual amorphoussolid was washed with benzene, set aside in the air overnight, andstirred with N aqueous sodium hydroxide (0.5 ml.) to give a suspensionof colourless fine needles. The crystals were collected, washed with afew drops of N aqueous sodium hydroxide, and dried in the air to givethe sodium salt of 9-deoxy-6,9α-epoxy-Δ⁵ -prostaglandin F₁α. Theinhibition of arachidonic acid-induced aggregation of human platelets ata conentration of 0.2 ng./ml. by this salt and its instability in waterat acid pH, together with further evidence, is compatible withassignation of the configuration(5Z)-5,6-didehydro-9-deoxy-6,9α-epoxyprostaglandin F₁α sodium salt.

The high-resolution ¹³ C n.m.r. spectrum of a solution of the crystalsin dimethyl sulphoxide-d₆ showed the expected 20 resonances whosechemical shifts were entirely consistent with the chemical structureestablished for Prostacyclin. No impurity peaks were detected.

EXAMPLE 7

5ξ-Iodo-9-deoxy-6ξ,9α-epoxyprostaglandin F₁α methyl ester (500 mg) wasstirred with methanolic NaOMe prepared from Na (0.23 g., 10 equivs.) andMeOH (3.5 ml.) under N₂ at room temperature overnight; 1N aq. NaOH (2.5ml.) was added to the yellow reaction solution to bring about hydrolysisof the ester moiety and, after 2 hours, the methanol was evaporated invacuo at room temperature. The residual aqueous solution gave risespontaneously to a mass of colourless fine needles of the desired sodiumsalt (formula (I): R=Na) which was cooled (0°), collected, washedsparingly with 1N aq. NaOH, air-dried, and stored in a stoppered tube;this salt (383 mg.) had νmax (KBr disc) 1692 cm⁻¹ ##STR4## and twenty ¹³C resonances only were observed (at 182.7(C-1), 158.2(C-6), 140.0 and134.3(C-13,14), 100.7(C-5), 87.5(C-15), 80.6 and 75.5(C-9,11),50.8(C-12), 49.0, 45.8, 42.4, 41.9, 37.5, 35.8(C-18), 31.6., 29.9, 29.3,26.7(C-19), and 18.4(C-20) ppm from TMS in DMSO-d₆). The product, sodium(5Z)-5,6-didehydro-9-deoxy-6,9α-epoxyprostaglandin F₁α (syn. sodiumprostacyclin), thus obtained complete inhibited arachidonic acid-inducedplatelet aggregation (human platelet-rich plasma) at 1 ng./ml. and itsprofile of biological activity on the rabbit aorta, rabbit coeliacartery, rat stomach strip and rat colon conformed with that of sodiumprostacyclin obtained by biosynthesis. After air-drying, the salt has asurface coating of sodium carbonate (ca 3.5% by weight) which protectsthe vinyl ether moiety against carbondioxide catalysed hydrolysis.

EXAMPLE 8

5ξ-Iodo-9-deoxy-6ξ,9α-epoxyprostaglandin F₁α methyl ester was treatedwith 1,5-diazabicyclo-5-nonene (DBN) at room temperature in the absenceof a solvent for a few hours. The DBN and hydrogen iodide wereconveniently removed by adsorption on to a column of SiO₂, prepared froma suspension of SiO₂ in EtOAc/Et₃ N 50:1, and the vinyl ether was elutedwith the same solvent system. I.R. spectroscopy (thin film, _(v) max1738 (CO₂ Me) and 1696 cm⁻¹ ##STR5## ¹ H n.m.r. in C₆ D₆ -Et₃ N, 19:1(δ4.22, triplet of triplets¹², J6.9 and 1.0 Hz (C-5 vinyl proton)), and¹³ C n.m.r. in C₆ D₆ -Et₃ N, 19:1 (distinctive features were resonancesat 159.8 (C-1), 155.8 (C-6), 137.2 and 130.6 (C-13,14), 95.3 (C-5), 84.1(C-15) 77.3 and 72.2 (C-9,11) and 51.1. (Me ester) p.p.m. from TMS).

The vinyl ether (5Z)-5,6-didehydro-9-deoxy-6,9α-epoxyprostaglandinmethyl ester, was hydrolysed with aqueous sodium hydroxide to givesodium prostacyclin (formula (I): R=sodium).

What we claim is:
 1. A method of treating a body portion for transplantwhich comprises contacting said body portion with prostacyclin.
 2. Amethod of preserving an isolated body portion, which comprisescontacting said body portion with prostacyclin.
 3. A method of perfusingor providing circulation for an isolated body portion with a fluid whichcomprises contacting said isolated body with fluid containingprostacyclin anion.
 4. The method of claim 1 in which said prostacyclinis in blood.
 5. The method of claim 2 in which said prostacyclin is inblood.
 6. The method of claim 3 in which the fluid is blood or a productthereof.